Low‐intensity low‐frequency ultrasound mediates riboflavin delivery during corneal crosslinking

Abstract We employed the mechanical effect from 40 kHz ultrasound (US) to improve the delivery of riboflavin into corneal stroma for collagen crosslinking, which can benefit the treatment of keratoconus and other corneal ectasias. Experiments were conducted, first with porcine corneas ex vivo and then with New Zealand white rabbits in vivo, at varying mechanical index (MI) and sonication time. Results showed that 15 min of US applied on the cornea at MI = 0.8 in the presence of 0.5% of riboflavin solution enabled its delivery to deeper corneal stroma. Excessive heat was removed by a cooling setup to negate the thermal effect. The corneal absorption amount and penetration of riboflavin through cornea as detected by fluorotron, as well as the enhancement of corneal stiffness as measured by Young's modulus, were comparable to the conventional approach that requires complete corneal epithelium debridement. Histological analysis revealed minor exfoliation of superficial cell layers of corneal epithelium and loss of ZO‐1 tight junctions immediately after US. Full recovery of the corneal epithelium and restoration of tight junctions occurred in 3–4 days. The study shows that low‐intensity low‐frequency ultrasound (LILF US) is a less invasive alternative to the conventional epithelium‐off method for delivering riboflavin into the corneal stroma.

mainly attributed to the removal of epithelium, which is necessary for absorption of riboflavin into the corneal stroma. 4,5 Removal of corneal epithelium necessitates the use of contact lens in the postoperative period, which further increases the risk of infectious keratitis in the postoperative period.
Riboflavin is used in C-CXL as photosensitizer within the stromal layer to promote electron transfer and generation of reactive oxygen species for CXL. 2 Nonetheless, the corneal epithelium poses as a physical, hydrophobic barrier to delivery of riboflavin to the stromal layer. The diffusion of riboflavin via the paracellular route is largely attenuated by the tight junctions within the stratified corneal epithelial cells. Furthermore, the transcellular route is limited with the scarce riboflavin transporters present on corneal surface. 6 Therefore, C-CXL involves de-epithelization to permit rapid and sufficient accumulation of riboflavin in the stromal layer before UV exposure. 7 However, removal of corneal epithelium has been associated with complications such as infectious keratitis, dry eye disease, and corneal haze. [8][9][10] Several strategies to bypass the need of de-epithelization procedure have been developed. They involve modifying the riboflavin formulations, employing less invasive physical approaches such as electric current or ultrasound (US). Delivery enhancers such as benzalkonium chloride, ethylenediaminetetraacetic acid (EDTA), and hydrophobic polymers have been added in riboflavin formulations to improve the corneal permeability to the small hydrophilic drugs.
However, these methods require longer instillation time to permit sufficient penetration of riboflavin across the stromal region. In contrast, iontophoresis utilizes electric current to drive the electroosmosis of charged riboflavin across the corneal barriers in significantly shorter time. Nonetheless, the corneal stiffening results were still sub-optimal to C-CXL. 11,12 On the other hand, US is another type of physical mode of delivery that has been widely adopted to improve drug penetration across the epithelial barriers of various organ sites, such skin, brain, and eyes. 13,14 This method enjoys compliance among the patients and practitioners owing to its safety and capacity for spatiotemporally controlled delivery of agents to the targeted areas. [15][16][17] US can exert nonthermal (mechanical) and thermal effects on the tissue barriers. The nonthermal effects mainly come from acoustic streaming and cavitation. The increase in emitted US intensity could help to promote the nonthermal effects for drug delivery. Nonetheless, this could lead to concurrent elevation of thermal energy, which may exert damaging effect on the tissues. 18 Of noted, US at the low-intensity and low-frequency mode (LILF-US) has the ability to circumvent the concurrent thermal effects. Based on the definition of mechanical index (MI), it was suggested that the low-frequency regime can permit greater increase in US intensity to give greater mechanical energy that facilitates drug delivery across the tissue barrier. 19,20 Under LF regime, Zderic et al. applied US at 880 kHz and 0.19-0.56 W/cm 2 on ex vivo rabbit cornea and reported an improvement of 2.1-4.2 folds in permeability of sodium fluorescein. 21 Lamy et al. increased the US of energy while keeping the same frequency (1 W/cm 2 at 880 kHz) to enhance riboflavin penetration to the cornea stroma and achieved 2.6-3.0 folds higher riboflavin concentration at 250 μm depth. 22 In addition, Nabili utilized continuous US at 400 kHz, 0.8 W/cm 2 and delivered dexamethasone across cornea into aqueous humor in 5 min on rabbit in vivo. 23 Previously, our research group applied US at much lower frequency and energy (40 kHz at 0.1 W/cm 2 ) for transscleral delivery. Using such low-frequency, low-intensity pulsed US wave, 90 s for thee cycles, macromolecules were delivered to the intraocular space, primarily by acoustic cavitation-bubble dynamics generated by the US wave. 24 We hypothesized that the same US frequency (40 kHz), which maximize the cavitation effect, would also facilitate trans-corneal penetration of oxygen and hydrophilic molecules, for example, the riboflavin. Oxygen and riboflavin are two of the principal components in CXL. In the meantime, we also installed a cooling system to remove the heat dissipation to minimize the thermal effect.
This research studied the effect of several parameters related to US mediation on the corneal permeability to riboflavin, including MI, dwell time, and concentration of riboflavin solution. The abovementioned parameters were optimized with porcine eyes, then tested on live rabbit eyes in vivo to evaluate enhancement of corneal absorption of riboflavin and improvement of corneal stiffness after UV treatment. The study also probed the safety of US method by monitoring the recovery of corneal surface on live rabbit eyes and examining the barrier histology of corneal epithelium post-US treatment.

| Riboflavin solution
Riboflavin 5 0 -monophosphate sodium salt hydrate (F2253; Sigma-Aldrich) was dissolved in phosphate-buffered saline (1x PBS) to a final concentration of 0.1% and 0.5% (w/v) and adjusted to pH 7. The riboflavin solution was aerated with compressed air for 10 min immediately before US experiment.

| US device assembly and calibration
The US device assembly consisted of a signal function generator

| Cornea adaptor for US application to the eye
A corneal adaptor was modified from a standard confocal dish (made of polystyrene with glass bottom) for ex vivo experiments of porcine eye using porcine eyes (Figure 1a). An orifice of 12 mm was made in the dish to expose the central cornea region of the porcine eyeball to the riboflavin solution loaded inside the cornea adaptor ( Figure S2).
The adaptor positions the corneal surface at 16 mm away from the US transducer surface (which is at the near field distance of the transducer in the current setup). Cooling to the adaptor was provided by a circulating cold-water bath in the surrounding. During the US experiment, the temperature rise of the solution inside the adaptor was maintained within 1 C, as confirmed by the thermocouple placed near the cornea surface ( Figure S3).
For in vivo experiments with live rabbits, an orifice of 10 mm was made to allow fitting of the smaller rabbit eye (Figure 1b). A soft silicone material was added to the top of the orifice to minimize mechanical damage of the cornea surface. A wearable strap was added to the adaptor to stabilize it to the rabbit eye. A sealable side inlet was added to allow filling of the riboflavin solution. During the experiment, the rabbit laid on its side such that the treated eye faced downwards to the adaptor. The adaptor was placed on top of the transducer, and the distance between corneal surface and the transducer surface was 16 mm (same as the ex vivo experiment). A cooling system similar to that in the ex vivo experiment was set up. Noncontact digital infrared thermometer was also used to detect the temperature of cornea surface during the US experiment. The temperature fluctuation was kept within 1 C with reference to the co-lateral.

| Ex vivo experiments using porcine eyes
Fresh porcine eyes were obtained from a local slaughterhouse within 2-5 h postmortem. The corneal surface was visually examined with a slit lamp. Porcine eyes with damaged cornea were excluded, and those with intact epithelium were randomly assigned into different groups, immediately used for ex vivo experiments. Batch-to-batch variation was controlled by comparing the riboflavin adsorption data of epi-on groups.
To prepare eyes in Epi Off control group (epithelium debridement), the corneal surface was soaked with 20% (v/v) ethanol for 20 s and the whole area of corneal epithelium was scraped off using a blunt hockey blade. 3 The details of the treatment groups are summarized in Table 1. Varying duration and MI were investigated in the UStreated groups. MI was calculated as follows: where P r is the rarefaction peak pressure (in MPa, as detected by hydrophone) and f is the frequency (in kHz) of US. 25 In all the conditions tested, the dwell time of riboflavin solution was kept at 30 min.

| In vivo experiments using rabbits
New Zealand white (NZW) rabbits (3.0-3.5 kg, 12 months old) were used. All animals were housed in HKUST Laboratory Animal Facility and all animal experiments had been approved by the HKUST Animal Ethics Committee. Rabbits were assigned at random to different experimental groups (Table 2). Using the set-up as illustrated in In the Epi-on group, the rabbit cornea remained intact and was exposed to the riboflavin solution for 30 min. In the control groups, the same set-up as Figure 1b was used for these control groups except that no US was applied.
F I G U R E 1 Schematic illustration of the ultrasound experiment setup. (a) In ex vivo experiment, porcine eye was placed on the corneal adaptor with an orifice of 12 mm. The ultrasound probe was directly under the adaptor and the distance to the corneal surface was 16 mm. The adaptor was filled with riboflavin solution. The adaptor was placed in a running cold-water bath to minimize the thermal effect from sonication. (b) In in vivo experiment, orifice opening lined with soft material and was changed to 10 mm. Rabbit was placed on its side, with eye facing down the adaptor filled with riboflavin solution. A similar cooling system was installed to minimize any thermal effect from sonication.
To investigate ocular biocompatibility, one eye of the rabbit  T A B L E 1 Treatment groups in ex vivo study using porcine eyes to reveal the effect of varying MI and treatment time for ultrasound-mediated delivery of riboflavin delivery 2.8 | Measurement of mechanical property of corneal strips The strain was then increased linearly with a velocity of 1.5 mm/min, and the stress was measured up to 2 Â 10 5 Pa. 3 The stress-strain curve was fitted to an exponential function σ ¼ Aexp B Á ε ð Þ using a software (SPSS GmbH Software). Young's modulus (E) was calculated at 10% strain as the gradient of the mean stress-strain graphs (E ¼ dσ=dε).  corresponds to the middle of the stromal region of cornea. 27 The results support that US not only enables riboflavin to cross the epithelium barrier but also promotes its penetration to deeper cornea.

| Evaluation of ocular biocompatibility
After confirming that US at 40 kHz MI = 0.8 was the most effective among the MI studied for increasing corneal absorption and penetration by riboflavin, the duration of sonication was investigated. In the current clinical practice, patient's cornea is exposed to riboflavin for 30 min after the corneal epithelium is stripped off. 3  Young's modulus was 14% and 36% higher than that of the non-CXL group when US was continuously applied for 20 and 30 min, respectively, whereas no statistically significant increase was observed when the duration was shortened to 10 min (Figure 4b). Also, there was no change in the stiffness of the corneas in the negative control group (Epi-on, no US, CXL), supporting that without the assistance of US in delivery, intact epithelium blocked riboflavin penetration.  Histological analysis revealed that the cellular structure of corneal epithelium at the superficial region was disrupted at the time point immediately after US treatment compared with naive rabbit cornea without any treatment (Figure 9 and Figure S6). However, the disruption was limited to the superficial cells and wing cells. The basal cell layer, which is the posterior-most layer of the corneal epithelium was intact. According to study on corneal basement, 8

| DISCUSSION
The potential advantages of using US for drug, gene, and protein delivery have been reported by us and others. 21,24,28,29 In these stud-  method (with the corneal epithelium barrier scraped off). 21,23 The total amount of riboflavin delivered to the cornea, its penetration into deeper cornea, and most importantly, the increase in the corneal stiffness upon CXL were comparable.
Although our focus is to employ the mechanical effect from US, heat generation from sonication is inevitable. Hyperthermia can denature proteins, leading to changes in pathways, membrane dysfunction, apoptosis, necrosis, clonogenic effects (interferences with mitosis). 18,31 Since the eye consists of delicate structures, it is especially important to minimize any thermal damage. FDA specifies that a medical device applied on the eye should produce a thermal index (TI) less than one, meaning that the temperature rise should be kept below 1 C. 32 We addressed this problem by installing a cooling system around the corneal adaptor ( Figure 1). An extended duration of sonication to allow sufficient mechanical abrasion on the corneal barrier would not be possible without the elimination of excessive heat.
For the CXL procedure to be an effective treatment for keratoconus, an optimal riboflavin concentration in the corneal stroma is required, as determined by a theoretical model previously. 21 Riboflavin acts as a photosensitizer that creates free radicals which, on UV irradiation, lead to the formation of new chemical bonds to increase CXL and hence the corneal stiffness. 20  writingreview and editing (lead).